Skip to main content
SpringerLink
Log in

A synthetic cadmium metallothionein gene (PMCd1syn) of Paramecium species: expression, purification and characteristics of metallothionein protein

  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

Metallothioneins (MTs) are metal binding proteins that are rich in cysteine residues constituting 10–30 % of the total protein, and in which the thiol groups bind to the metal ions. The increasing amount of metal ions in the medium have shown increased production of MTs by different organisms such as bacteria, protozoa and mammals like humans. PMCd1 is the first gene ever discovered in Paramecium, a ciliated protozoan, that could produce this MT in response to cadmium. In this study the PMCd1syn gene has been cloned in pET41a expression vector and expressed in an Escherichia coli BL21-codonplus strain for the first time. Since the gene PMCd1 amplified from Paramecium contained 10 codons, which could act as stop codons during expression in E. coli, this gene of 612 bps was synthesized to substitute these (stop) codons for the Paramecium sp. specific amino acids. For stability of the expressed protein, glutathione-S-transferase gene was fused with PMCd1syn gene and coexpressed. The cells expressing PMCd1syn demonstrated increased accumulation of cadmium. This is the first report of cadmium MT protein expressed from Paramecium species, particularly from synthetic MT gene (PMCd1syn). This fusion protein, the molecular weight of which has been confirmed to be 53.03 kDa with MALDI analysis, is rich in cysteine residues, and has been shown for the first time in this ciliate to bind to and sequester Cd2+-ions.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11

Similar content being viewed by others

Abbreviations

MT:

Metallothionein

Cd:

Cadmium

PMCd1 :

Cadmium-binding MT gene from Paramecium species

PMCd1syn :

Synthetic PMCd1 gene

C:

Cysteine

GST:

Glutathione-S-transferase

References

  1. Kägi JH, Kojima Y (1987) Chemistry and biochemistry of metallothionein. Experientia Suppl 52:25–61

    PubMed  Google Scholar 

  2. Kagi JH (1991) Overview of metallothionein. Meth Enzymol 205:613–626

    Article  PubMed  CAS  Google Scholar 

  3. Coyle P, Philcox JC, Carey LC, Rofe AM (2002) Metallothionein: the multipurpose protein. Cell Mol Life 59:627–647

    Article  CAS  Google Scholar 

  4. Vasak M, Hasler DW, Faller P (2000) Metal thiolate clusters in neuronal growth inhibitor factor (GIF). J Inorg Biochem 79:7–10

    Article  PubMed  CAS  Google Scholar 

  5. Capdevila M, Atrian S (2011) Metallothionein protein evolution: an miniassay. J Biol Inorg Chem 16:977–989

    Article  PubMed  CAS  Google Scholar 

  6. Henkel G, Krebs B (2004) MTs: zinc, cadmium, mercury, and copper thiolates and selenolates mimicking protein active site features-structural aspects and biological implications. Chem Rev 104:801–824

    Article  PubMed  CAS  Google Scholar 

  7. Isart NR, Vasak M (2002) Advances in structure and chemistry of metallothioneins. Inorg Biochem 88:388–396

    Article  Google Scholar 

  8. Arseniev A, Schultze P, Wörgötter E, Braun W, Wagner G, Vasák M, Kägi JH, Wüthrich K (1988) Three-dimensional structure of rabbit liver [Cd7] metallothionein-2a in aqueous solution determined by nuclear magnetic resonance. J Mol Biol 201:637–657

    Article  PubMed  CAS  Google Scholar 

  9. Braun W, Vasak M, Robbins AH, Stout CD, Wagner G, Kagi JHR, Wuthrich K (1992) Polypeptide fold into metal clusters of metallothionein 2 by nuclear magnetic resonance in solution. Proc natl Acad Sci USA 89:10124–10128

    Article  PubMed  CAS  Google Scholar 

  10. Peterson CW, Narula SS, Armitage IM (1996) 3D solution structure of copper and silver-substituted yeast metallothioneins. Fed Europ Biochem Soc 379:85–93

    Article  CAS  Google Scholar 

  11. Riek R, Prêcheur B, Wang Y, Mackay EA, Wider G, Güntert P, Liu A, Kägi JH, Wüthrich K (1999) NMR structure of the sea urchin, Strongylocentrotus purpuratus metallothionein MTA. J Mol Biol 291:417–428

    Article  PubMed  CAS  Google Scholar 

  12. Zangger K, Oz G, Otvos JD, Armitage IM (1999) Three-dimensional solution structure of mouse [Cd7]-metallothionein-1 by homonuclear and heteronuclear NMR spectroscopy. Protein Sci 8:2630–2638

    Article  PubMed  CAS  Google Scholar 

  13. Blindauer CA, Sadler PJ (2005) How to hide zinc in a small protein. Acc Chem Res 38:62–69

    Article  PubMed  CAS  Google Scholar 

  14. Bertini I, Hartmann HJ, Klein T, Liu GH, Luchinat C, Weser U (2000) High resolution solution structure of the protein part of Cu7 metallothionein. Eur J Biochem 267:1008–1018

    Article  PubMed  CAS  Google Scholar 

  15. Oz G, Zangger K, Armitage IM (2001) Three-dimensional structure and dynamics of a brain specific growth inhibitory factor: metallothionein-3. Biochemistry 40:11433–11441

    Article  PubMed  CAS  Google Scholar 

  16. Munoz A, Forsterling FH, Shaw CF, Petering DH (2002) Structure of the Cd-113(3) beta domains from Homarus americanus metallothionein-1: hydrogen bonding and solvent accessibility of sulfur atoms. J Biol Inorg Chem 7:713–724

    Article  PubMed  CAS  Google Scholar 

  17. Capasso C, Carginale V, Crescenzi O, Di Maro D, Parisi E, Spadaccini R, Temussi PA (2003) Solution structure of MT_nc, a novel metallothionein from the Antarctic fish Notothenia coriiceps. Structure 11:435–443

    Article  PubMed  CAS  Google Scholar 

  18. Cobine PA, McKay RT, Zangger K, Dameron CT, Armitage IM (2004) Solution structure of Cu6 metallothionein from the fungus Neurospora. Eur J Biochem 271:4213–4221

    Article  PubMed  CAS  Google Scholar 

  19. Calderone V, Dolderer B, Hartmann HJ, Echner H, Luchinat C, Del Bianco C, Mangani S, Weser U (2005) The crystal structure of yeast copper thionein: the solution of a long-lasting enigma. Proc Natl Acad Sci USA 102:51–56

    Article  PubMed  CAS  Google Scholar 

  20. Wang H, Zhang Q, Cai B, Li HY, Sze KH, Huang ZX, Wu HM, Sun HZ (2006) The molecular structure and vibrational spectra of corrolazine metal complexes, CzM by density functional theory. FEBS J 580:795–800

    Article  CAS  Google Scholar 

  21. Peroza EA, Schmucki R, Guntert P, Freisinger E, Zerbe O (2009) The beta(E)-domain of wheat E(c)1 metallothionein: a metal-binding domain with a distinctive structure. J Mol Biol 387:207–218

    Article  PubMed  CAS  Google Scholar 

  22. Namdarghanbari MA, Meeusen J, Bachowski G, Giebel N, Johnson J, Petering DH (2010) Reaction of the zinc sensor FluoZin-3 with Zn7-metallothionein: inquiry into the existence of a proposed weak binding site. J Inorg Biochem 104:224–231

    Article  PubMed  CAS  Google Scholar 

  23. Zhang L, Wu J, Wang X, Liu B, Ma B (2012) Isolation of metallothionein genes and in silico structural characterization of their proteins using molecular modeling from Yak (Bos grunniens). Biochem Genet. doi:10.1007/s10528-012-9503-7

    Google Scholar 

  24. Robinson NJ, Whitehall SK, Cavet JS (2001) Microbial metallothioneins. Adv Microb Physiol 44:183–213

    Article  PubMed  CAS  Google Scholar 

  25. Sabolić I, Breljak D, Skarica M, Herak-Kramberger CM (2010) Role of MT in cadmium traffic and toxicity in kidneys and other mammalian organs. Biometals 23:897–926

    Article  PubMed  Google Scholar 

  26. Meyer JN, Lord CA, Yang XY, Turner EA, Badireddy AR, Marinakos SM, Chilkoti A, Wiesner MR, Auffan M (2010) Intracellular uptake and associated toxicity of silver nanoparticles in Caenorhabditis elegans. Aquat Toxicol 100:140–150

    Article  PubMed  CAS  Google Scholar 

  27. Egli D, Yepiskoposyan H, Selvaraj A, Balamurugan K, Rajaram R (2006) A family knockout of all four Drosophila MTs reveals a central role in copper homeostasis and detoxification. Mol Cell Biol 26:2286–2296

    Article  PubMed  CAS  Google Scholar 

  28. Hughes S, Sturzenbaum SR (2007) Single and double MT knockout in the nematode C. elegans reveals cadmium dependent and independent toxic effects on life history traits. Environ Pollut 145:395–400

    Article  PubMed  CAS  Google Scholar 

  29. Thirumoorthy N, Sunder AS, Kumar KTM, Kumar MS, Ganesh, GNK, Chatterjee M (2011) A review of metallothionein isoforms and their role in pathophysiology. World J Surg Oncol 9:54. http://www.wjso.com/content/9/1/54

    Google Scholar 

  30. Diaz S, Amaro F, Rico D, Campos V, Benitez L, Martin-Gonzalez A, Hamilton EP, Orias E, Gutierrez JC (2007) Tetrahymena metallothioneins fall into two discrete subfamilies. PLoS ONE 2(3)e291 doi:10.1371/journal.pone.0000291

  31. Wang Q, Xu J, Chai B, Liang A, Wang W (2011) Functional comparison of metallothionein MTT1 and MTT2 from Tetrahymena thermophile. Arch Biochem Biophys 509:170–176

    Article  PubMed  CAS  Google Scholar 

  32. Fu C, Miao W (2006) Cloning and characterization of a new multi-stress inducible metallothionein gene in Tetrahymena pyriformis. Protist 157:193–203

    Article  PubMed  CAS  Google Scholar 

  33. Piccinni E, Bertaggia D, Santovito G, Miceli C, Kraen A (1999) Cadmium metallothionein gene of Tetrahymena pyriformis. Gene 234:51–59

    Article  PubMed  CAS  Google Scholar 

  34. Dondero F, Cavaletto M, Chezzi AR, La Terza A, Banni M, Viarengo A (2004) Biochemical characterization and quantitative gene expression analysis of the multi-stress inducible metallothionein from Tetrahymena thermophila. Protist 155:157–168

    Article  PubMed  CAS  Google Scholar 

  35. Shang Y, Song X, Bowen J, Corstanje R, Gao Y, Gaertig J, Gorovsky MA (2002) A robust inducible-repressible promoter greatly facilitates gene knockouts, conditional expression, and overexpression of homologous and heterologous genes in Tetrahymena thermophila. Proc Natl Acad Sci USA 99:3734–3739

    Article  PubMed  CAS  Google Scholar 

  36. Shuja RN, Shakoori AR (2007) Identification, cloning and sequencing of a novel stress inducible metallothionein gene from locally isolated Tetrahymena tropicalis lahorensis. Gene 4905:19–26

    Article  Google Scholar 

  37. Amaro F, de Lucas MDP, Martin-Gonzalez A, Gutierrez JC (2008) Two new members of the Tetrahymena multi-stress inducible metallothionein family: characterization and expression analysis of T. rostrata Cd/Cu metallothionein genes. Gene 423:85–91

    Article  PubMed  CAS  Google Scholar 

  38. Boldrin F, Santovito G, Formigari A, Bisharyan Y, Cassidy-Hanley D, Clark TG, Piccini E (2008) MTT2, a copper inducible metallothionein gene from Tetrahymena thermophila. Comp Biochem Physiol C: Toxicol Pharmacol 147:232–240

    Article  Google Scholar 

  39. Boldrin F, Santovito G, Irato P, Piccini E (2002) Metal interaction and regulation of Tertrahymena pigmentosa metallothionein genes. Protist 153:283–291

    Article  PubMed  CAS  Google Scholar 

  40. Hinrichsen RD, Tran JR (2010) A circadian clock regulates sensitivity to cadmium in Paramecium tetraaurelia. Cell Biol Toxicol 26:379–389

    Article  PubMed  CAS  Google Scholar 

  41. Shuja RN, Shakoori AR (2009) Identification and cloning of first cadmium metallothionein like gene from locally isolated ciliate, Paramecium sp. Mol Biol Rep 36:549–560

    Article  PubMed  CAS  Google Scholar 

  42. Sambrook J, Russel DW (2001) Molecular cloning: a labotratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  43. Sanger F, Coulson AR, Barrell GG, Smith AJH, Roe BA (1980) Cloning in single stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol 143:161–178

    Article  PubMed  CAS  Google Scholar 

  44. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc natl Acad Sci USA 74:5463–5467

    Article  PubMed  CAS  Google Scholar 

  45. Ma Y, Lin J, Zhang C, Ren Y, Lin J (2011) Cd(II) and AS(III) bioaccumulation by recombinant Escherichia coli expressing oligomeric human metallothioneins. J Haz Mat 185:1605–1608

    Article  CAS  Google Scholar 

  46. Li Y, Cockburn W, Kilpatrick J, Whitelam GC (2000) Cytoplasmic expression of a soluble synthetic mammalian metallothionein-α domain in Escherichia coli: enhanced tolerance and accumulation of cadmium. Mol. Biotech 16:211–219

    Article  Google Scholar 

  47. Berka T, Shatzman A, Zimmerman J, Strickler JM, Rosenberg M (1988) Efficient expression of the yeast metallothionein gene in Escherichia coli. J Bact 170:210–226

    Google Scholar 

  48. Sayers Z, Brouillon P, Vorgias CE, Nolting HF, Hermes C, Koch MH (1993) Cloning and expression of Saccharomyces cerevisiae copper metallothionein gene in Escherichia coli and characterization of recombinant protein. Eur J Biochem 212:521–528

    Article  PubMed  CAS  Google Scholar 

  49. Murooka Y, Nagaoka T (1987) Expression of cloned monkey metallothionein in Escherichia coli. Appl Environ Microbiol 53:204–207

    PubMed  CAS  Google Scholar 

  50. Hou YM, Kim R, Kim SH (1988) Expression of the mouse metallothionein -1 gene in Escherichia coli: increased tolerance to heavy metals. Biochim Biophys Acta 951:230–234

    Article  PubMed  CAS  Google Scholar 

  51. Kille P, Stephens P, Cryer A, KLay J (1990) The expression of a synthetic rainbow trout metallothionein gene in E. coli. Biochim Biophys Acta 1048:178–186

    Article  PubMed  CAS  Google Scholar 

  52. Odawara F, Kurasaki M, Suzuki-Kurasaki M, Oikawa S, Emoto T, Yamasaki F, Linde Arias AR, Kojima Y (1995) Expression of human metallothionein-2 in Escherichia coli: cadmium tolerance of transformed cells. J Biochem 118:1131–1137

    PubMed  CAS  Google Scholar 

  53. Gutierrez JC, Amaro F, Diaz S, de Francisco P, Cubas LL, Martin-Gonzalez A (2011) Ciliate metallothioneins: unique microbial eukaryotic heavy-metal binder molecules. J Biol Inorg Chem 16:1025–1034

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

ARS acknowledges with appreciation the partial financial support provided by Pakistan Academy of Sciences for this project.

Author information

Authors and Affiliations

  1. School of Biological Sciences, University of the Punjab, Quaid-e-Azam Campus, Lahore, 54590, Pakistan

    Saira Dar, Rukhsana N. Shuja & Abdul Rauf Shakoori

Authors
  1. Saira Dar
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rukhsana N. Shuja
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Abdul Rauf Shakoori
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Abdul Rauf Shakoori.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Dar, S., Shuja, R.N. & Shakoori, A.R. A synthetic cadmium metallothionein gene (PMCd1syn) of Paramecium species: expression, purification and characteristics of metallothionein protein. Mol Biol Rep 40, 983–997 (2013). https://doi.org/10.1007/s11033-012-2140-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-012-2140-8

Keywords

Search

Navigation